The clinical treatment most frequently used for the revascularization of vessels affected by arteriosclerosis is transluminal percutaneous coronary angioplasty (PCTA). A pathological process frequently associated to this intervention is restenosis, consisting of the excessive reocclusion of the operated vessel. Restenosis has a high healthcare and socioeconomic impact, since it makes it necessary to repeat the PCTA or subjecting the patient affected to alternative revascularization therapies (for example, aortocoronary by-pass). In comparison with the native atheromatous lesion, characterized by a slow development (typically over decades), the restenotic lesion usually grows during the first 4-12 months after the PCTA (Serruys, Kutryk and Ong, “Coronary-artery stents”, N Engl J Med 2006, 354, 483-95). Currently, over 90% of PCTA use support metal endoprostheses called stents which increase the safety of the procedure and have reduced restenosis rates to 15-30%, compared with rates of 25-50% typically associated to conventional PCTA (Serruys, Kutryk and Ong, “Coronary-artery stents”, N Engl J Med 2006, 354, 483-95, Andrés, “Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential”, Cardiovasc Res 2004, 63, 11-21). Restenosis rates are further reduced with the use of drug-eluting stents.
Restenosis is a multifactorial process in which various cell types intervene, mainly platelets, monocytes/macrophages, endothelial cells (ECs), and smooth muscle cells (SMCs). It is accepted that the growth of the restenotic lesion, also called neointimal lesion, is a process started by the mechanical damage that causes the implantation of the stent (Andrés, “Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential” Cardiovasc Res 2004, 63, 11-21, Costa and Simon, “Molecular basis of restenosis and drug-eluting stents”, Circulation 2005, 111, 2257-73). The initial acute phase of restenosis involves the activation of platelets and localized thrombosis, accompanied by the recruitment of circulating monocytes, neutrophils and lymphocytes on the damaged arterial surface. These cell types unleash a chronic inflammatory response characterized by the activation of the SMCs resident in the tunica media, which adopt a “synthetic” phenotype characterized by morphological changes, expression of embryonic isoform of contractile proteins, high responsiveness to growth and chemotactic stimulus, and abundant synthesis of extracellular matrix. A plethora of chemotactic and mitogenic factors produced by the cells of the neointimal lesion causes a first proliferative phase of the SMCs of the tunica media and their migration towards the lesion, followed by a second hyperplastic response of the SMCs of the neointimal lesion (Andrés, “Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential”, Cardiovasc Res 2004, 63, 11-21, Costa and Simon, “Molecular basis of restenosis and drug-eluting stents” Circulation 2005, 111, 2257-73). The resolution of the inflammation and cicatrization of the vascular lesion in later stages of the PCTA goes accompanied by the restoration of the contractile phenotype of neointimal SMCs and changes in the composition of the extracellular matrix which becomes more similar to the undamaged arterial wall. As previously indicated, if the restenosis is excessive, the clinical symptoms reappear making a further revascularization intervention necessary.
Among the neointimal hyperplasia regulators identified in animal and human studies, the following are included: thrombogenic factors (for example, tissue factor, thrombin receptor), cell adhesion molecules (for example, VCAM, ICAM, LFA-1, Mac-1), signal transducers (for example, PI3K, MEK/ERK), transcription factors (for example, NF-κB, E2F, AP-1, c-myc, c-myb, YY1, Gax), cell cycle regulatory proteins (for example, pRb, p21, p27, CDK2, CDC2, cyclin B1, PCNA), growth factors (for example, PDGF-BB, TGFβ, FGF, IGF, EGF, VEGF), inflammatory cytokines (for example, TNFα), chemotactic factors (for example, CCR2, MCP-1), and metalloproteases (for example, MMP-2, MMP-9).
The essentially hyperproliferat ive character of restenosis has generated great interest in the study of the role that cell cycle regulatory genes may play in this pathological process. In mammals, the cell cycle is regulated positively by holoenzymes composed of a catalytic subunit called cyclin-dependent kinase (CDK) and a regulatory subunit called cyclin (Ekholm and Reed, “Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle”, Curr Opin Cell Biol 2000, 12, 676-84). The sequential activation of the CDK/cyclins permits different events of phosphorylation of cell substrates involved in cell proliferation. On the other hand, there are inhibitory proteins of CDKs/cyclins called CKIs (CDK inhibitors), which are subdivided into the CIP/KIP (p21, p27 and p57) and INK4 (p15, p16, p18, and p19) subfamilies. The accumulation of CKIs in response to anti-mitogenic stimuli provokes the reversible inhibition of CDK/cyclin complexes. Expression studies and gene therapy experiments have revealed the importance of these molecules in the development of the neointimal lesion. Thus, the analysis of obstructive vascular lesions induced by mechanical damage in animal and human models of angioplasty has demonstrated alterations in the expression of cell cycle regulatory genes (for example, CDKs, cyclins, CKIs, p53, pRb), and numerous experimental animal studies have shown that the inactivation of CDKs and cyclins (for example, cyclin B, CDK2, CDK1), or the overexpression of growth suppressors (for example p21, p27, pRb, p53) inhibits the development of obstructive vascular lesions after angioplasty (Andrés, “Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential”, Cardiovasc Res 2004, 63, 11-21, Nabel, “CDKs and CKIs: molecular targets for tissue remodelling” Nat Rev Drug Discov 2002, 1, 587-98, Dzau, Braun-Dullaeus and Sedding, “Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies”, Nat Med 2002, 8, 1249-56).
Numerous systemic therapeutic approaches to prevent or treat restenosis failed in clinical trials despite encouraging preclinical data derived from various animal models. However, the recent introduction of the antiproliferative drug-eluting stents (DES) has revolutionized interventional cardiology. We should highlight the use of stents to deliver sirolimus (also called rapamycin or rapamune) and paclitaxel (also called taxol), two lipophilic drugs which have as target the common final route of cell proliferation, the mitotic cycle of the eukaryotic cell. The use of these devices, which locally release high doses of the drug in the damaged arterial wall, has significantly reduced restenosis rates (Costa and Simon, “Molecular basis of restenosis and drug-eluting stents”, Circulation 2005, 111, 2257-73, Wessely, Schomig and Kastrati, “Sirolimus and Paclitaxel on polymer-based drug-eluting stents: similar but different”, J Am Coll Cardiol 2006, 47, 708-14). For this reason, 2 DESs are implanted out of every 3 stents currently implanted in Europe (Baz, Mauri, Albarran and Pinar, “[Spanish Cardiac Catheterization and Coronary Intervention Registry. 16th Official Report of the Spanish Society of Cardiology Working Group on Cardiac Catheterization and Interventional Cardiology (1990-2006)]”, Rev Esp Cardiol 2007, 60, 1273-89). Relevant drawbacks of the use of DES with respect to conventional stents are their high cost (2-3 times more) and the need to prolong the anti-platelet treatment to avoid adverse events associated to late thrombosis (reviewed in Lazaro and de Mercado, “Stents recubiertos de fármacos: eficacia, efectividad, eficiencia y evidencia”, Revista Española de Cardiología 2004, 57, 608-12).
Due to the high healthcare and socio-economic impact of restenosis, it would be highly useful to have biomarkers that could be quantified in a reproducible, reliable and cost effective form in patients needing revascularization. The possibility of estimating the risk of restenosis in these patients could help in taking therapeutic decisions, for example, implantation of stents versus aortocoronary by-pass, or use of conventional stents versus DES (more expensive and with an increased risk of late thrombosis).
Single-nucleotide polymorphisms (SNP) are genetic variants present by millions throughout the human genome. In recent years SNPs have been identified in human genes which are associated with a greater or lesser risk of developing restenosis, including the gene of the beta2-adrenergic receptor, CD14, colony stimulating factor (CSF), eotaxin, caspase-1, P2RY12 receptor, and interleukin-10 (Monraats et al. “Inflammation and apoptosis genes and the risk of restenosis after percutaneous coronary intervention”, Pharmacogenet Genomics 2006, 16, 747-754; Monraats et al. “Interleukin 10: a new risk marker for the development of restenosis after percutaneous coronary intervention”, Genes Immun 2007, 8, 44-50; Monraats et al. “Genetic inflammatory factors predict restenosis after percutaneous coronary interventions”, Circulation 2005, 112, 2417-25; Rudez et al. “Platelet receptor P2RY12 haplotypes predict restenosis after percutaneous coronary interventions”, Hum Mutat 2008, 29, 375-80).
However, no genotype-phenotype associations have been described to date relating SNPs in cell cycle regulatory genes with a greater or lesser risk of developing restenosis.
The authors of the present invention, after important research work, have identified different SNPs in various cell cycle regulatory genes with potential diagnostic value as genetic risk markers for developing restenosis. Specifically, they have identified the SNPs rs164390, rs350099, rs350104, rs875459, in the CCNB1 gene (cyclin B1 protein); rs2282411, in the CCNA1 gene (cyclin A1 protein) and rs733590, in the CDKN1A gene (p21Kip1/Cip1 protein) as markers of the diagnosis of the risk of developing restenosis.
These markers constitute a very important advance in the taking of therapeutic decisions. For example, patients with relative low risk of developing restenosis could receive a conventional stent, whilst the use of DES (more expensive and with a greater risk of late thrombosis) could be limited to patients with greater risk.
Based on these findings, the authors of the invention have developed a method to determine the risk of restenosis after the implantation of a stent based on the detection of these 6 SNPs as diagnostic markers of said risk. Likewise, they have developed a kit to carry out said diagnosis.